Compositions for delivering bisphosphonates

ABSTRACT

Compounds and compositions for the delivery of bisphosphonates are provided. Methods of preparation, administration and treatment are provided as well.

This application claims the benefit of U.S. Provisional Application No.60/272,676, filed Mar. 1, 2001, which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to compounds and compositions fordelivering bisphosphonates to a target. These compounds are well suitedfor forming non-covalent mixtures with bisphosphonates for oraladministration to animals. Methods for preparation, administration andtreatment are also disclosed.

BACKGROUND OF THE INVENTION

Conventional means for delivering active agents are often severelylimited by biological, chemical, and physical barriers. Typically, thesebarriers are imposed by the environment through which delivery occurs,the environment of the target for delivery, and/or the target itself.Biologically and chemically active agents are particularly vulnerable tosuch barriers.

In the delivery to animals of biologically active and chemically activepharmacological and therapeutic agents, barriers are imposed by thebody. Examples of physical barriers are the skin, lipid bi-layers andvarious organ membranes that are relatively impermeable to certainactive agents but must be traversed before reaching a target, such asthe circulatory system. Chemical barriers include, but are not limitedto, pH variations in the gastrointestinal (GI) tract and degradingenzymes.

These barriers are of particular significance in the design of oraldelivery systems. Oral delivery of many biologically or chemicallyactive agents would be the route of choice for administration to animalsif not for biological, chemical, and physical barriers that prevent,restrict or reduce the passage of active agents. Among the numerousagents in this category are bisphosphonates. Bisphosphonates areroutinely prescribed for the treatment and/or prevention ofosteoporosis. (See Drug Delivery Today, Yates, A. John and Rodan, Gideon“Alendronate and Osteoporosis” vol 3 No.; 2 pgs 69-78 February, 1998)Although some bisphosphonates are currently available in oral tabletdosage forms, the mean oral bioavailability relative to an intravenous(IV) reference dose is low; for example, alendronate has a reported meanbioavailability of 0.7% for doses ranging from 5 to 40 mg whenadministered after an overnight fast. Earlier methods for orallyadministering vulnerable pharmacological agents have relied on theco-administration of adjuvants (e.g., resorcinols and non-ionicsurfactants such as polyoxyethylene oleyl ether andn-hexadecylpolyethylene ether) to increase artificially the permeabilityof the intestinal walls, as well as the co-administration of enzymaticinhibitors (e.g., pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation. Liposomes have also been described as drug delivery systemsfor insulin and heparin. However, broad spectrum use of such drugdelivery systems is precluded because: (1) the systems require toxicamounts of adjuvants or inhibitors; (2) suitable low molecular weightcargos, i.e. active agents, are not available; (3) the systems exhibitpoor stability and inadequate shelf life; (4) the systems are difficultto manufacture; (5) the systems fail to protect the active agent(cargo); (6) the systems adversely alter the active agent; or (7) thesystems fail to allow or promote absorption of the active agent.

Certain modified amino acids have been used to deliver pharmaceuticals.See, for example, U.S. Pat. Nos. 5,629,020; 5,643,957; 5,650,386;5,766,633; 5,776,888; and 5,866,536; and PCT application WO00/06534.

There is a need for simple, inexpensive delivery systems which areeasily prepared for the delivery of bisphosphonates.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising at least one ofthe delivery agent compounds of the following formulas and at least onebisphosphonate. These compositions facilitate the delivery of thebisphosphonate to selected biological systems and increase or improvethe bioavailability of bisphosphonate compared to administration withoutthe delivery agent compound. Delivery agent compounds of the presentinvention include those having the following formulas or salts thereof:

In another preferred embodiment, the composition comprises abisphosponate and a delivery agent of the following structure and saltsthereof:

wherein Ar is phenyl or naphthyl; optionally substituted with OH,halogen, C₁-C₄ alkyl, C₁-C₄ alkenyl, C₁-C₄ alkoxy or C₁-C₄ haloalkoxy;

R⁷ is selected from the group consisting of C₄-C₂₀ alkyl, C₄-C₂₀alkenyl, phenyl, naphthyl, (C₁-C₁₀ alkyl) phenyl, (C₁-C₁₀alkenyl)phenyl, (C₁-C₁₀ alkyl) naphthyl, (C₁-C₁₀ alkenyl) naphthyl,phenyl (C₁-C₁₀ alkyl), phenyl (C₁-C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl),or naphthyl (C₁-C₁₀ alkenyl);

R⁸ is selected from the group consisting of hydrogen, C₁ to C₄ alkyl, C₂to C₄ alkenyl, C₁ to C₄ alkoxy, C₁-C₄ and haloalkoxy;

R⁷ is optionally substituted with C₁ to C₄ alkyl, C₂ to C₄ alkenyl, C₁to C₄ alkoxy, C₁-C₄ haloalkoxy, —OH, —SH, and —CO₂R⁹ or any combinationthereof;

R⁹ is hydrogen, C₁ to C₄ alkyl or C₂ to C₄ alkenyl.

R⁷ is optionally interrupted by oxygen, nitrogen, sulfur or anycombination thereof;

with the proviso that the compounds are not substituted with an aminogroup in the position alpha to the acid group or salts thereof.

According to one preferred embodiment, Ar is substituted with an OH.According another preferred embodiment, Ar is substituted with OH andhalogen.

Preferably, R⁷ is C₄-C₂₀ alkyl or phenyl (C₁-C₁₀ alkyl) More preferablyR⁷ is C₅-C₁₀ alkyl or phenyl (C₂ alkyl). Most preferably, R⁷ is C₇-C₉alkyl or phenyl(C₂ alkyl).

Preferred carrier compounds are of the formulas of Compounds 1, 2, 3, 4or salts thereof.

In another embodiment, the composition comprises a bisphosphonate and adelivery agent of the following structure and salts thereof:

wherein

-   -   Ar is phenyl or naphthyl;    -   Ar is optionally substituted with C₁-C₄ alkyl, C₁-C₄ alkoxy,        C₂-C₄ alkenyl, C₂-C₄ alkynyl, aryl, aryloxy, a heterocyclic        ring, C₅-C₇ carbocylic ring, halogen, —OH, —SH, CO₂R⁶, —NR⁷R⁸,        or —N⁺R⁷R⁸R⁹Y⁻;

-   (a) R¹ is C₁-C₁₆ alkylene, C₂-C₁₆ alkenylene, C₂-C₁₆ alkynylene,    C₆-C₁₆ arylene, (C₁-C₁₆ alkyl) arylene, or aryl (C₁-C₁₆ alkylene);    -   R² is —NR³R⁴ or —N⁺R³R⁴R⁵Y⁻;

R³ and R⁴ are independently hydrogen; oxygen; hydroxy; substituted orunsubstituted C₁-C₁₆ alkyl; substituted or unsubstituted C₂-C₁₆ alkenyl;substituted or unsubstituted C₂-C₁₆ alkynyl; substituted orunsubstituted aryl; substituted or unsubstituted alkylcarbonyl;substituted or unsubstituted arylcarbonyl; substituted or unsubstitutedalkanesulfinyl; substituted or unsubstituted arylsulfinyl; substitutedor unsubstituted alkanesulfonyl; substituted or unsubstitutedarylsulfonyl; substituted or unsubstituted alkoxycarbonyl; substitutedor unsubstituted aryloxycarbonyl;

R⁵ is independently hydrogen; substituted or unsubstituted C₁-C₁₆ alkyl;substituted or unsubstituted C₂-C₁₆ alkenyl; substituted orunsubstituted C₂-C₁₆ alkynyl; substituted or unsubstituted aryl;substituted or unsubstituted alkylcarbonyl; substituted or unsubstitutedarylcarbonyl; substituted or unsubstituted alkanesulfinyl; substitutedor unsubstituted arylsulfinyl; substituted or unsubstitutedalkanesulfonyl; substituted or unsubstituted arylsulfonyl; substitutedor unsubstituted alkoxycarbonyl; substituted or unsubstitutedaryloxycarbonyl;

-   -   (b) R¹, R², and R⁵ are as defined above; and

R³and R⁴ are combined to form a 5, 6 or 7-membered heterocyclic ring; or5, 6 or 7-membered heterocyclic ring substituted with a C₁-C₆ alkyl,C₁-C₆ alkoxy, aryl, aryloxy, oxo group or carbocyclic ring; or

-   -   (c) R² and R⁵ are as defined above; and

R¹ and R³are combined to form a 5, 6 or 7-membered heterocyclic ring; or5, 6 or 7-membered heterocyclic ring substituted with a C₁-C₆ alkyl,alkoxy, aryl, aryloxy, or oxo group or carbocyclic ring;

R⁴ is hydrogen; oxygen; hydroxy; substituted or unsubstituted C₁-C₁₆alkyl; substituted or unsubstituted C₂-C₁₆ alkenyl; substituted orunsubstituted C₂-C₁₆ alkynyl; substituted or unsubstituted aryl;substituted or unsubstituted alkylcarbonyl; substituted or unsubstitutedarylcarbonyl; substituted or unsubstituted alkanesulfinyl; substitutedor unsubstituted arylsulfinyl; substituted or unsubstitutedalkanesulfonyl; substituted or unsubstituted arylsulfonyl; substitutedor unsubstituted alkoxycarbonyl; substituted or unsubstitutedaryloxycarbonyl;

R⁶ is hydrogen; C₁-C₄ alkyl; C₁-C₄ alkyl substituted halogen or —OH;C₂-C₄ alkenyl; or C₂-C₄ alkenyl substituted halogen or —OH;

R⁷, R⁸, and R⁹ are independently hydrogen; oxygen; C₁-C₄ alkyl; C₁-C₄alkyl substituted with halogen or —OH; C₂-C₄ alkenyl; or C₂-C₄alkenylsubstituted with halogen or —OH; and

Y is halogen, hydroxide, sulfate, nitrate, phosphate, alkoxy,perchlorate, tetrafluoroborate, or caboxylate. A non-limiting example ofa suitable carboxylate is acetate.

The term “substituted” as used herein with respect to compound Bincludes, but is not limited to, the following substituents: halogen and—OH.

In one preferred embodiment, Ar is unsubstituted phenyl or phenylsubstituted with one or more of C₁-C₄ alkyl, C₁-C₄ alkoxy, or halogen;more preferably the phenyl is substituted with methoxy, Cl, F or Br.;more preferably the substitution is Cl.

In another preferred embodiment, R¹ is C₁-C₁₂ alkyl, more preferablyC₂-C₈ alkyl, more preferably C₂-C₆ alkyl, and more preferably C₆ alkyl.

In another preferred embodiment, R³ and R⁴ are independently H or C₁-C₂alkyl; more preferably R³ and R⁴ are not both H; more preferably R³ andR⁴ are independently methyl or ethyl; and more preferably R³ and R⁴ areboth methyl.

In another preferred embodiment, the compound has the formula ofCompound 5 or salts thereof.

Also provided are dosage unit forms comprising the compositions. Thedosage unit may be in the form of a liquid or a solid, such as a tablet,capsule or particle, including a powder or sachet.

Another embodiment is a method for administering a bisphosphonate to ananimal in need thereof, by administering a composition of the presentinvention to the animal. The preferred route of administration is oral.

Yet another embodiment is a method of treating and/or preventingbone-related disorders in an animal by administering the composition ofthe present invention to the animal. Typically, an effective amount ofthe composition is administered to treat and/or prevent the desiredbone-related disorder.

Yet another embodiment is a method of preparing a composition of thepresent invention by mixing at least one delivery agent compound and atleast one bisphosphonate.

DETAILED DESCRIPTION OF THE INVENTION

Delivery Agent Compounds

The terms “alkyl” and “alkenyl” as used herein include linear andbranched alkyl and alkenyl substituents, respectively.

The delivery agent compounds depicted as carboxylic acids may be in theform of the carboxylic acid or salts thereof. Suitable salts include,but are not limited to, organic and inorganic salts, for examplealkali-metal salts, such as sodium (e.g., monosodium and disodium salts,such as monosodium and disodium salts of compounds 1-4 and 7-9),potassium and lithium; alkaline-earth metal salts, such as magnesium,calcium or barium; ammonium salts; basic amino acids, such as lysine orarginine; and organic amines, such as dimethylamine or pyridine.Preferably, the salts are sodium salts. The salts may be mono- ormulti-valent salts, such as monosodium salts and di-sodium salts. Thesalts may also be solvates, including ethanol solvates, and hydrates.

The delivery agent compounds depicted as amines may be in the form ofthe free amine or salts thereof. Suitable salts include, but are notlimited to, organic and inorganic salts, for example hydrochloridesalts, acetate or citrate.

Salts of the delivery agent compounds of the present invention may beprepared by methods known in the art. For example, sodium salts may beprepared by dissolving the delivery agent compound in ethanol and addingaqueous sodium hydroxide.

Where the delivery agent has an amine moiety and a carboxylic acidmoiety, poly amino acids and peptides comprising one or more of thesecompounds may be used. An amino acid is any carboxylic acid having atleast one free amine group and includes naturally occurring andsynthetic amino acids. Poly amino acids are either peptides (which aretwo or more amino acids joined by a peptide bond) or are two or moreamino acids linked by a bond formed by other groups which can be linkedby, e.g., an ester or an anhydride linkage. Peptides can vary in lengthfrom dipeptides with two amino acids to polypeptides with severalhundred amino acids.

One or more of the amino acids or peptide units may be acylated orsulfonated.

The compounds described herein may be derived from amino acids and canbe readily prepared from amino acids by methods within the skill ofthose in the art, such as those described in WO96/30036, WO97/36480,WO00/06534, WO00/46812, WO00/50386, WO00/59863, WO 01/32596, WO00/07979, U.S. Pat. No. 5,643,957, U.S. Pat. No. 5,650,386, and U.S.Pat. No. 5,866,536, all of which are incorporated by reference. Forexample, the compounds may be prepared by reacting the single amino acidwith the appropriate acylating or amine-modifying agent, which reactswith a free amino moiety present in the amino acid to form amides.Protecting groups may be used to avoid unwanted side reactions as wouldbe known to those skilled in the art. With regard to protecting groups,reference is made to T. W. Greene, Protecting Groups in OrganicSynthesis, Wiley, N.Y. (1981), the disclosure of which is herebyincorporated herein by reference.

The delivery agent compound may be purified by recrystallization or byfractionation on one or more solid chromatographic supports, alone orlinked in tandem. Suitable recrystallization solvent systems include,but are not limited to, acetonitrile, methanol, ethanol, ethyl acetate,heptane, water, tetrahydrofuran, and combinations thereof.

Fractionation may be performed on a suitable chromatographic supportsuch as alumina, using methanol/n-propanol mixtures as the mobile phase;reverse phase chromatography using trifluoroacetic acid/acetonitrilemixtures as the mobile phase; and ion exchange chromatography usingwater or an appropriate buffer as the mobile phase. When anion exchangechromatography is performed, preferably a 0-500 mM sodium chloridegradient is employed.

Bisphosphonates

The term bisphosphonates refers to pyrophosphate analogs in which thecentral oxygen of the phosphorous-oxygen-phosphorous portion of themolecule, is replaced with a carbon to give aphosphorous-carbon-phosphorous moiety. Examples of bisphosphonatesinclude but are not limited to alendronate, clodronate, etidronate,ibandronate, incadronate, minodronate, neridronate, olpadronate,pamidronate, risedronate, tiludronate, zoledronate, EB1053, YH529, andany analogs, mimetics, and polyethylene glycol-modified derivativesthereof.

Delivery Systems

The composition of the present invention comprises one or more deliveryagent compounds and one or more bisphosphonate. In one embodiment, oneor more of the delivery agent compounds is mixed with one or morebisphosphonates prior to administration to form an administrationcomposition.

The administration compositions may be in the form of a liquid. Thesolution medium may be water. Dosing solutions may be prepared by mixinga solution of the delivery agent compound with a solution of thebisphosphonate, just prior to administration. Alternately, a solution ofthe delivery agent compound (or bisphosphonate) may be mixed with thesolid form of the bisphosphonate (or delivery agent compound). Thedelivery agent compound and the bisphosphonate may also be mixed as drypowders. The delivery agent compound and the bisphosphonate can also beadmixed during the manufacturing process.

The dosing solutions may optionally contain additives such as phosphatebuffer salts, citric acid, glycols, or other dispersing agents.Stabilizing additives may be incorporated into the solution, preferablyat a concentration ranging between about 0.1 and 20% (w/v)

The administration compositions may alternately be in the form of asolid, such as a tablet, capsule or particle, such as a powder orsachet. Solid dosage forms may be prepared by mixing the solid form ofthe compound with the solid form of the bisphosphonate. Alternately, asolid may be obtained from a solution of delivery agent compound andbisphosphonate by methods known in the art, such as freeze-drying(lyophilization), precipitation, crystallization and solid dispersion.

The administration compositions of the present invention may alsoinclude one or more enzyme inhibitors. Such enzyme inhibitors include,but are not limited to, compounds such as actinonin or epiactinonin andderivatives thereof. Other enzyme inhibitors include, but are notlimited to, aprotinin (Trasylol) and Bowman-Birk inhibitor.

The amount of bisphosphonate used in an administration composition ofthe present invention is an amount effective to accomplish the purposeof the bisphosphonate for the target indication. The amount ofbisphosphonate in the compositions typically is a pharmacologically,biologically, therapeutically, or chemically effective amount. However,the amount can be less than that amount when the composition is used ina dosage unit form because the dosage unit form may contain a pluralityof delivery agent compound/bisphosphonate compositions or may contain adivided pharmacologically, biologically, therapeutically, or chemicallyeffective amount.

The total effective amount can then be administered in cumulative unitscontaining, in total, an effective amount of bisphosphonate.

The total amount of bisphosphonate to be used can be determined bymethods known to those skilled in the art. However, because thecompositions of the invention may deliver bisphosphonate moreefficiently than compositions containing the bisphosphonate alone, loweramounts of bisphosphonate than those used in prior dosage unit forms ordelivery systems can be administered to the subject, while stillachieving the same blood levels and/or therapeutic effects.

The presently disclosed delivery agent compounds facilitate the deliveryof bisphosphonate, particularly in oral form, but may also be useful inintranasal, sublingual, intraduodenal, subcutaneous, buccal,intracolonic, rectal, vaginal, mucosal, pulmonary, transdermal,intradermal, parenteral, intravenous, intramuscular and ocular systems.

Dosage unit forms can also include any one or combination of excipients,diluents, disintegrants, lubricants, plasticizers, colorants,flavorants, taste-masking agents, sugars, sweeteners, salts, and dosingvehicles, including, but not limited to, water, 1,2-propane diol,ethanol, olive oil, or any combination thereof.

The compounds and compositions of the subject invention are useful foradministering bisphosphonates to any animals, including but not limitedto birds such as chickens; and mammals, such as rodents, cows, pigs,dogs, cats, primates, and particularly humans.

The system is particularly advantageous for delivering bisphosphonatesthat would otherwise be destroyed or rendered less effective byconditions encountered before the bisphosphonate reaches its target zone(i.e. the area in which the bisphosphonate is to be released) and withinthe body of the animal to which they are administered. Particularly, thecompounds and compositions of the present invention are useful in orallyadministering bisphosphonates, especially those that are not ordinarilyorally deliverable, or those for which improved delivery is desired.

The compositions of the present invention have utility in the deliveryof bisphosphonates to selected biological systems and in increasingand/or improving the bioavailability of bisphosphonates compared toadministration of bisphosphonates alone. Delivery and/or bioavailabilitycan be increased and/or improved by delivering more of thebisphosphonate over a period of time, or in delivering morebisphosphonate at a specific time, or in delivering bisphosphonate in aparticular time period (such as to effect quicker or delayed delivery)or in delivering bisphosphonate over a period of time (such as sustaineddelivery).

The composition of the present invention can be administered to treatand/or prevent any disease for which bisphosphonates are known to becapable of treating and/or preventing. Typically, an effective amount ofthe composition is administered to treat and/or prevent the desireddisease.

Another embodiment of the present invention is a method for thetreatment and/or prevention of bone-related disorders in an animal byadministering the composition of the present invention to the animal.Typically, an effective amount of the composition is administered totreat and/or prevent the desired bone-related disorder. Bone-relateddisorders include, but are not limited to, disorders of the bone anddisease states, and include but are not limited to osteoporosis, bonedegeneration, Paget's disease, and/or osteoclast function (for example,inhibiting osteoclasts).

Specific indications for bisphosphonate can be found in the Physicians'Desk Reference (54^(th) Ed., 2000, Medical Economics Company, Inc.,Montvale, N.J.), which is herein incorporated by reference.

The appropriate amount of the bisphosphonate and delivery agent can bedetermined by methods known in the art.

Following administration, the bisphosphonate is taken up into thecirculation. The bioavailability of the bisphosphonates is calculatedfrom its excretion in the urine or in its uptake in bone, according tomethods used in the art.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples illustrate the invention without limitation. Allparts are given by weight unless otherwise indicated.

Proton nuclear magnetic resonance (¹H NMR) analyses for the compoundslisted below were conducted on a 300 MHz Bruker spectrometer usingdimethyl sulfoxide (DMSO-d₆) as the solvent unless otherwise indicated.

EXAMPLE 1 Compound Preparation

Preparation of Compound 1

Sodium N-salicyloyl-8-aminocaprylate

The sodium salt of Compound 1 may be prepared according to the methodsof U.S. Pat. No. 5,650,386, WO00/46182 or WO00/59863.

Preparation of Compound 2

10-(N-salicyloylamino)decanoic acid

Compound 2 may be prepared according to the methods of U.S. Pat. No.5,866,536, WO00/46182 or WO00/59863.

Preparation of Compound 3

9-(salicyloylamino)nonanoic acid

Compound 3 may be prepared according to the methods of U.S. Pat. No.5,866,536, WO00/46182 or WO00/59863 using the appropriate startingmaterials.

Preparation of Compound 4

Preparation of 2-(4-(N-salicyloyl)aminophenyl)propionic acid

Compound 4 may be made by the following method:

A slurry of 58.6 g (0.355 mol) of 2-(4-aminophenyl) propionic acid and500 mL of methylene chloride was treated with 90.11 mL (77.13 g, 0.710mol) of trimethylsilyl chloride and was heated to reflux for 120 min.The reaction mixture was cooled to 0° C. and treated with 184.44 mL(107.77 g, 1.065 mol) of triethylamine. After stirring for 5 minutes,this mixture was treated with a solution of 70.45 g (0.355 mol) ofO-acetylsalicyloyl chloride and 150 mL of methylene chloride.

The reaction mixture was warmed to 25° C. and stirred for 64 hours. Thevolatiles were removed in vacuo. The residue was stirred in 2N aqueoussodium hydroxide for one hour and acidified with 2M aqueous sulfuricacid. The solid was recrystallized twice from ethanol/water to give atan solid. Isolation by filtration afforded 53.05 g of (52% yield) of2-(4-(N-salicyloyl) aminophenyl) propionic acid. Solubility: 200 mg/mL(200 mg+350 μl 2N NaOH+650 μl H₂O, pH=7.67). Anal. Calculated: C=67.36,H=5.3, N=4.91. Found: C=67.05, H=5.25, N=4.72.

Preparation of Compound 4 Sodium Salt

Preparation of Sodium 2-(4-(N-salicyloyl)aminophenyl)propionate

The sodium salt of compound 4 may be made by the following method:

A solution of 53.05 g (0.186 mol) of 2-(4-(N-salicyloyl)aminophenyl)propionic acid and 300 mL of ethanol was treated with 7.59 g (0.190 mol)of NaOH dissolved in 22 mL of water. The reaction mixture was stirredfor 30 minutes at 25° C. and for 30 minutes at 0° C. The resulting paleyellow solid was isolated by filtration to give 52.61 g of sodium2-(4-(N-salicyloyl) aminophenyl) propionate. Solubility: 200 mg/mL clearsolution, pH=6.85. Anal. Calculated: C=60.45, H=5.45, N=3.92, Na=6.43.Found: C=60.84, H=5.87, N=3.85, Na=6.43. Melting point 236-238° C.

Preparation of Compound 5

N-(6-Dimethylaminohexyl)salicylamide

Compound 5 may be made by the following method:

A slurry of 18.02 g (110 mmol) of carsalam, 18.0 mL (15.84 g, 109 mmol)of 6-dimethylamino-1-hexanol, 29.12g (111 mmol) of triphenylphosphine,.and 150 mL of tetrahydrofuran was treated with a solution of 21.8 mL(22.39 g, 111 mmol) of diisopropyl azodicarboxylate and 40 mL oftetrahydrofuran, added dropwise over 20 minutes, causing the temperatureof the slurry to rise to about 67° C. The reaction mixture was allowedto cool back to about 25° C. and stir for about 20 hours. The solutionwas treated with 150 mL (300 mmol) of aqueous 2N sodium hydroxide andwarmed to about 60° C. for about 90 minutes. The reaction mixture waswashed with ethyl acetate (2×60 mL). The aqueous phase was acidifiedwith 4% aqueous hydrochloric acid to a pH slightly less than about 0 andwashed with ethyl acetate (2×60 mL). The pH of the aqueous phase wasraised to about 4.5 with 50% aqueous potassium carbonate and washed withethyl acetate (2×60 mL). The aqueous phase was treated with solid sodiumbicarbonate and extracted with ethyl acetate (14×60 mL). The combined 14ethyl acetate extracts were dried over sodium sulfate and concentratedto a viscous liquid. The liquid was taken up into a minimum amount ofethyl acetate, diluted with 100 ml of hexanes and treated in an ice bathwith 150 mL of hexanes, causing a white solid to develop. A total of13.65 g of N-(6-dimethylaminohexyl)salicylamide was isolated byfiltration.

Preparation of Compound 6

8-(2-Acetylphenoxy)octanoic acid

Compound 6 may be prepared by the following method.

Potassium hydroxide (10.72 g, 191.1 mmol) was ground in a mortar untilpowdered, then added to a 250 mL round bottom flask containing 80 mL ofdimethyl sulfoxide. The resulting mixture was stirred for 5 minutes,after which time 6.47 g (47.5 mmol) of 2-hydroxyacetophenone was added,immediately followed by 24.04 g (95.7 mmol) of ethyl 8-bromooctanoate.The reaction was stirred at room temperature for one hour. The orangereaction mixture was poured into 200 mL of distilled water, thenextracted five times with 300 mL (total) of methylene chloride. Theorganic layers were washed with two 50 mL portions of water, thenconcentrated to give a bright yellow liquid.

The liquid was dissolved in 25 mL of dioxane. Aqueous sodium hydroxide(1N, 20 mL) was added, and the resulting liquid was stirred and heated(65° C.) for two hours. The reaction mixture was cooled to 0° C.,acidified to pH 1 with concentrated aqueous hydrochloric acid, thenextracted with two 100 mL portions of ethyl acetate. The organic layerwas concentrated to give a bright yellow oil. The oil was crystallizedwith methanol:water (1:1), then recrystallized once with methanol:water(1:1), and once with methylene chloride:hexanes (1:4), to give 5.70 g(43.1%) of a pale yellow to off white solid. Melting point: 71.5-73.5°C. Combustion analysis: % C: 69.04 (calc'd), 68.77 (found); % H: 7.97(calc'd), 8.04 (found). ¹H NMR Analysis: (d₆-DMSO):δ 12.0, s, 1H; 7.57,dd, 1H; 7.52, dt, 1H; 7.15, d, 1H; 7.00, dt, 1H; 4.09, t, 2H; 2.52, s,3H; 2.20, t, 2H; 1.78, p, 2H; 1.46, m, 4H; 1.32, m, 4H.

Preparation of Compound 7

8-(2-Hydroxyphenoxy)octanoic acid

Compound 7 may be prepared by the following method.

A 200 mL round bottom flask was charged with 22.9 g (3 equiv.) offreshly ground potassium hydroxide and 100 mL of dimethyl sulfoxide.This mixture was stirred at 25° C. for 5 minutes. Catechol (15 g, 1equiv.) was added followed immediately by ethyl 8-bromooctanoate (34.2g, 1 equiv). This dark brown solution was then stirred at 25° C. for 2hours.

Distilled water (100 mL) was added and this solution was heated to 85°C. for 2 hours. The mixture was cooled, acidified to pH˜2 withconcentrated aqueous hydrochloric acid, and extracted with ethyl acetate(300 mL×2). The combined organics were dried over magnesium sulfate,filtered and the solvent evaporated. The crude material was purified bysilica gel chromatography using 30-60% ethyl acetate/hexanes as eluent.The desired product was collected and dried to give 6.6 g (19%) of8-(2-hydroxyphenoxy)octanoic acid as an off-white solid. Melting point:60-64° C. Combustion analysis: % C: 66.65 (calc'd), 66.65 (found); % H:7.99 (calc'd), 8.10 (found). ¹H NMR Analysis: (d₆-DMSO): δ 12.0 s, 1H;8.8, s, 1H; 6.90-6.86, m, 1H; 6.80-6.76, m, 3 H; 3.92, t, 2H; 2.21 t,2H; 1.75-1.66, m, 2H; 1.56-1.29, m, 8H.

Alternate Preparation of Compound 7

A 500 mL Erlenmeyer flask was charged with 28 g (4 equiv.) of powderedpotassium hydroxide and 400 mL of dimethyl sulfoxide. This mixture wasstirred at room temperature for 5 minutes. 2-Benzyloxyphenol (25 g, 1equiv.) was added and followed immediately by addition of ethyl8-bromooctanoate (37.6 g, 1.2 equiv). The resulting solution was stirredat room temperature for 2 hours.

The reaction mixture was poured into 200 mL of distilled water andheated to 80° C. for 3 hours. This mixture was then acidified withconcentrated aqueous hydrochloric acid to a pH of approximately 2. Anoff-white solid precipitated. This solid was isolated by vacuumfiltration and allowed to dry overnight at room temperature in vacuo.The material was then esterified by reacting the crude acid with 1L ofmethanol and 5 mL of sulfuric acid and subsequent heating to 80° C.overnight.

The mixture was cooled and extracted with ethyl acetate 3×400 mL, driedover magnesium sulfate, filtered and evaporated to give the methyl esterin quantitative yield.

The crude ester was then dissolved in 150 mL of ethanol and mixed with 1g of 10% palladium on activated carbon. This mixture was placed in theParr autoclave. The reaction vessel was then pressurized to 200 psi withhydrogen. The heterogeneous mixture was stirred at 50° C. for 18 hours.The palladium was filtered off and the filtrate concentrated to give thedebenzylated product.

The methyl ester was saponified using 10 g of sodium hydroxide, 400 mLof methanol, and 50 mL of water. The solution was heated to 80° C. forone hour, and then allowed to stir at ambient temperature overnight. Themethanol was evaporated. An additional 100 mL of water was added and theaqueous layer acidified with concentrated aqueous hydrochloric acid to apH of 2. The aqueous phase was then extracted with ethyl acetate, 3×300mL, dried and evaporated to give the target material. The crude materialwas then purified by silica gel chromatography using 30-60% ethylacetate/hexanes, as eluent, to give 22.24 g (71%) of8-(2-hydroxyphenoxy)octanoic acid as an off-white solid. Melting point:65-68° C. Combustion analysis: % C: 66.65 (calc'd), 66.98 (found); % H:7.99 (calc'd) 8.22 (found). ¹H NMR Analysis: (d₆-DMSO): δ 12.0, s, 1H;8.8, s, 1H; 6.90-6.87, m, 1H; 6.80-6.67, m, 3H; 3.94, t, 2H; 2.23, t,2H; 1.73, p, 2H; 1.53-1.29, m, 8H.

Preparation of Compound 8

4-Hydroxyphenyl-8-oxyoctanoic acid

Compound 8 may be made by the following method:

Potassium hydroxide (11.20 g, 200.0 mmol) was ground in a mortar untilpowdered, then added to a 0.5 L round bottom flask containing 90 mL ofDMSO. The resulting mixture was stirred for 5 minutes, after which time10.00 g (50.0 mmol) of 4-benzyloxyphenol were added, immediatelyfollowed by 12.55 g (50.0 mmol) of ethyl 8-bromooctanoate. The reactionwas stirred at room temperature for two and one half hours. The reactionmixture was poured into 200 mL of distilled water, and heating to refluxwas initiated. This was allowed to continue heating for three and onehalf hours. Heating of the reaction mixture was then discontinued andthe reaction mixture was allowed to come to room temperature overnight.Heating was restarted the following day when it was determined that thehydrolysis was incomplete. After an additional three-hour period ofheating, it was determined that the reaction was completed and heatingwas discontinued. When the reaction mixture had cooled to roomtemperature, it was acidified with 2N, HCl solution and the resultingsolid was isolated by filtration. The solid was allowed to dry undervacuum overnight. 17.96 g of the 4-benzyloxyphenyl-8-oxyoctanoic acidwas isolated.

This material was used as is for the next step. The4-benzyloxyphenyl-8-oxyoctanoic acid was placed into a 0.5 L roundbottomed flask with 120 mL of ethyl alcohol. The mixture was sparged for15 minutes with nitrogen before 10% palladium on activated carbon wasadded to the reaction mixture. The flask was then evacuated and aballoon containing hydrogen was placed atop the flask in a way that thecontents of the flask were kept under a hydrogen atmosphere. Thismixture was allowed to stir overnight at room temperature, and was thenfiltered through celite. Ethyl alcohol was removed in vacuo, yielding awhite solid which was first recrystallized from 90:10 ethyl alcohol:water and then was dissolved in 2N NaOH. This mixture was filtered andacidified with 2N HCl. The resulting white solid was isolated byfiltration and allowed to dry under vacuum. 2.12 g of the4-hydroxyphenyl-8-oxyoctanoic acid was isolated. Melting point: 97-100°C. Combustion analysis: % C: 66.67 (calc.), 66.43 (found); % H: 7.94(calc.), 7.80 (found). ¹H NMR Analysis: (d₆-DMSO): δ 12.0, s, 1H; 9.00,s, 1H; 6.63, m, 4H; 3.75, t, 2H; 2.15, t, 2H; 1.60, p, 2H; 1.45, p, 2H;1.20, m, 6H.

Preparation of Compound 10

N-(5-chlorosalicyloyl)-8-aminocaprylate

The disodium salt of Compound 10 may be prepared according to themethods of WO00/59863.

Example 2 Bisphosphonate Oral Delivery

Oral dosing (PO) compositions of delivery agent compound and alendronate(anhydrous, monosodium salt) in water were prepared. Typically 400 mg ofdelivery agent compound was added to 2.0 mL of water. When the deliveryagent compound had a carboxylic acid terminal, either the sodium salt ofthe compound was used or the free acid was converted to the sodium saltby stirring the resultant solution and adding one equivalent of sodiumhydroxide (10.0 N) and diluting with water. The solution was vortexed,then heated (about 37° C.) and sonicated. The pH was adjusted to about 7(7.0 to 8.5) with NaOH or HCl. Additional NaOH (for carboxylic acidterminated delivery agents) or HCl (for amine terminated compounds) wasadded as necessary to achieve uniform solubility, and the pH readjusted.Water was then added to bring the total volume to about 2.5 mL (variesdepending on solubility of the delivery agent compound). Alendronate (25μl) from a stock solution (made from 2.0 g sodium alendronate in 10 mldeionized water, pH adjusted to about 7.5 with 10N NaOH, vortexed andsonicated at 37° C. to obtain a clear solution, frozen and defrostedbefore use) was added to the solution. The final doses were 200 mg/kgdelivery agent compound (i.e. 200 mg delivery agent compound per kg ofbody weight) and 2.5 mg/kg alendronate, and the volume dose was 1.0mL/kg.

The typical dosing and sampling protocols were as follows. MaleSprague-Dawley rats weighing between 200-250 g were fasted for 24 hoursprior to dosing. For those experiments where the dosing was tonon-fasted rats the animals had access to food and water ad libitum. Adosing group of five animals was administered one of the dosingsolutions. For oral dosing, an 11 cm Rusch 8 French catheter was adaptedto a 1 mL syringe with a pipette tip. The syringe was filled with dosingsolution by drawing the solution through the catheter, which was thenwiped dry. The catheter was placed down the esophagus leaving 1 cm oftubing past the incisors. Solution was administered by pressing thesyringe plunger.

After dosing, the animals were housed singly in metabolism cages. Foodwas administered 45 minutes post-dosing. Water was available ad libitum.Urine collection commenced as soon as the animals were placed in theircages. Urine samples were taken at 14 hours post dose. The samples werestored on dry ice until sampled. Quantitation was by HPLC method foralendronate.

As a control, 0.1 mg/kg alendronate (from a 2 mL sterile water solutionmade from 120 μl of a solution made from the following: 3.33 mg sodiumalendronate, 4.91 mg sodium chloride USP reagent crystals; 10.3 mgsodium citrate USP, 2.88 mg citric acid USP, in 10 mL deionized water,pH about 5.0.) was injected intravenously through the tail vein withoutanesthesia. Urine samples were collected as above. The results areillustrated in Table 1. ALN=alendronate.

TABLE 1 Efficacy of the Oral Delivery of Alendronate Average Amt.Delivery Route Of ALN Agent of Excreted* Std. Range Compound #administration (ng) Dev. n Mean F⁺ (ng) (none) IV 928.62 374.67 5 100.0± 1.61  392.28-1345.08 (none) PO 110.96 35.44 10   0.48 ± 0.15 62.32-169.32 1 PO 6366.27 3269.43 5  27.42 ± 14.10  2632.77-10057.85 4PO 5496.56 2401.44 5  23.68 ± 10.34 2376.68-8856.76 5 PO 2559.11 1616.035 11.02 ± 6.96  786.99-4544.08 6 PO 3180.87 1857.81 5 13.70 ± 8.00 889.44-5367.03 7 PO 2748.21 1474.64 5 11.84 ± 6.35  850.64-4300.44 8 PO1448.01 892.86 5  6.23 ± 3.84  340.70-2578.03 ⁺Mean Bioavailabilityrelative to IV Reference Dose *[ALN] times total amount of urineexcreted 14 hours post-dose Note: Delivery Agent dose is 200 mg/kg Allanimals were fasted for 24 hours.

TABLE 2 Oral Dose-Response: Alendronate and Compound 1 Alendronatedose/Delivery Agent Average dose (mg/mg) ALN in Delivery per kg bodyUrine⁺ Std. Agent weight (ng) Dev. n* Range (ng) (none) 2.5 325.93 74.205 253.44-434.12 1  2.5/200 6093.18 4561.10 4  1097.56-11890.55 1 1.0/200 1584.53 1030.16 5  408.64-3227.36 1 0.50/200 1716.17 851.79 4 788.20-2746.00 1 0.25/200 806.44 541.43 3  349.96-1404.64 1 0.10/200538.65 237.93 4 307.16-843.08 *n = number of samples that had reportablevalues of [ALN] out of 5 ⁺[ALN] in ng/mL times total amount of urineexcreted 14 hours post dose All animals were fasted for 24 hours.

TABLE 3 Oral Dose Response: Alendronate and Compound 10 Alendronatedose/Delivery Agent dose (mg/mg) Average ALN Delivery per kg body inUrine⁺ Std. Agent weight (ng) Dev. n* Range (ng) (none) 5 188.42 68.9 5105-253 9   5/200 1712.2 343.9 4 1354-2607 9  2.5/200 1017.67 176.73 5 490-1474 9 1.25/200 659.67 123.56 5  374-1103 9 0.62/200 343-83 102.815  86-812 ⁺[ALN] in ng/mL times total amount of urine excreted 14 hourspost dose All animals were fasted for 24 hours

TABLE 4 Oral Administration of Alendronate: Effects of FastingAlendronate dose/Delivery Average Agent ALN Delivery dose (mg/mg) per inUrine⁺ Std. Fasted or Agent kg body weight (ng) Dev. n* non-Fasted 1 2.550.88 14.18 5 Fasted 1 2.5 12.32 5.8 5 non-Fasted 1 2.5/200 609 192.1 5Fasted 1 2.5/200 20.3 69.18 5 non-Fasted 9 2.5 47.88 14.56 5 Fasted 92.5 10.55 5.74 5 non-Fasted 9 2.5/200 1509 195.90 5 Fasted 9 2.5/200210.13 150.44 5 non-Fasted ⁺[ALN] in ng/mL times total amount of urineexcreted 14 hours post dose

TABLE 5 Delivery Agent Dose Response: Alendronate and Compound 10Alendronate dose/Delivery ALN Agent dose Average Delivery (mg/mg) per inStd. Agent kg body weight Urine⁺ (ng) Dev. n* 9 2.5/0 147 98.3 5 92.5/200 1140.67 191.98 5 9 2.5/100 473.33 320.81 5 9 2.5/50 418.33221.55 5 9 2.5/50 97 324.39 5 ⁺[ALN] in ng/mL times total amount ofurine excreted 14 hours post dose All animals were fasted for 24 hours

The above mentioned patents, applications, test methods, andpublications are hereby incorporated by reference in their entirety.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed description. Allsuch obvious variations are within the fully intended scope of theappended claims.

1. A composition comprising: (A) alendronate; and (B) at least one compound selected from

and salts thereof.
 2. The composition of claim 1 wherein (B) is

Compound 1 or a salt thereof.
 3. The composition of claim 2 wherein (B) is the sodium salt of compound
 1. 4. The composition of claim 1 wherein (B) is

Compound 2 or a salt thereof.
 5. The composition of claim 1 wherein (B) is

Compound 3 or a salt thereof.
 6. The composition of claim 1 wherein (B) is

Compound 4 or a salt thereof.
 7. The composition of claim 1 wherein (B) is

Compound 5 or a salt thereof.
 8. The composition of claim 1 wherein (B) is

Compound 6 or a salt thereof.
 9. The composition of claim 1 wherein (B) is

Compound 7 or a salt thereof.
 10. The composition of claim 1 wherein (B) is

Compound 8 or a salt thereof.
 11. A composition comprising: (A) ibandronate; and (B) at least one compound selected from

and salts thereof.
 12. The composition of claim 11 wherein (B) is

Compound 1 or a salt thereof.
 13. The composition of claim 12 wherein (B) is the sodium salt of compound
 1. 14. The composition of claim 11 wherein (B) is

Compound 2 or a salt thereof.
 15. The composition of claim 11 wherein (B) is

Compound 3 or a salt thereof.
 16. The composition of claim 11 wherein (B) is

Compound 4 or a salt thereof.
 17. The composition of claim 11 wherein (B) is

Compound 5 or a salt thereof.
 18. The composition of claim 11 wherein (B) is

Compound 6 or a salt thereof.
 19. The composition of claim 11 wherein (B) is

Compound 7 or a salt thereof.
 20. The composition of claim 11 wherein (B) is

Compound 8 or a salt thereof.
 21. A composition comprising: (A) risedronate; and (B) at least one compound selected from

and salts thereof.
 22. The composition of claim 21 wherein (B) is

Compound 1 or a salt thereof.
 23. The composition of claim 22 wherein (B) is the sodium salt of compound
 1. 24. The composition of claim 21 wherein (B) is

Compound 2 or a salt thereof.
 25. The composition of claim 21 wherein (B) is

Compound 3 or a salt thereof.
 26. The composition of claim 21 wherein (B) is

Compound 4 or a salt thereof.
 27. The composition of claim 21 wherein (B) is

Compound 5 or a salt thereof.
 28. The composition of claim 21 wherein (B) is

Compound 6 or a salt thereof.
 29. The composition of claim 21 wherein (B) is

Compound 7 or a salt thereof.
 30. The composition of claim 21 wherein (B) is

Compound 8 or a salt thereof.
 31. A composition comprising: (A) zoledronate; and (B) at least one compound selected from

and salts thereof.
 32. The composition of claim 31 wherein (B) is

Compound 1 or a salt thereof.
 33. The composition of claim 32 wherein (B) is the sodium salt of compound
 1. 34. The composition of claim 31 wherein (B) is

Compound 2 or a salt thereof.
 35. The composition of claim 31 wherein (B) is

Compound 3 or a salt thereof.
 36. The composition of claim 31 wherein (B) is

Compound 4 or a salt thereof.
 37. The composition of claim 31 wherein (B) is

Compound 5 or a salt thereof.
 38. The composition of claim 31 wherein (B) is

Compound 6 or a salt thereof.
 39. The composition of claim 31 wherein (B) is

Compound 7 or a salt thereof.
 40. The composition of claim 31 wherein (B) is

Compound 8 or a salt thereof. 